Non-woven mat and method of producing same

ABSTRACT

A mat having a highly uniform porosity distribution is produced by consolidating 15 or more layers of melt blown webs (or the like) having different orientations. Control over the porosity is provided by using webs that exhibit a narrow, unimodal distribution of fiber diameters over the bulk of its distribution, such as in the top 80%. A compliance of the mats can be chosen by selecting a number and orientation of the webs. It is thus possible to produce mats that are good candidates for vascular grafts, for example. The uniformity of the porosity within the range of 6 μm to 30 μm permits seeding of the vascular graft with endothelial and smooth muscle cells. The mats have the demonstrated ability to retain, and support growth of, smooth muscle cells and endothelial cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 12/452,231 filed Apr.22, 2010, the entire contents of which is herein incorporated byreference, which is a national entry of PCT/CA2008/000989 filed May 21,2008, which claims the benefit of United States Provisional PatentApplication U.S. Ser. No. 60/929,247 filed Jun. 19, 2007, the entirecontents of which are hereby incorporated by reference.

FIELD OF THE INVENTION

The invention relates in general to non-woven mats and to a novel methodof producing same, and, in particular, relates to a non-woven mat formedby pressing a plurality of differently oriented web sections of a webproduced, for example, by extrusion fiber melt blowing. The mats have auniformity of porosity and a compliance that makes it acceptable, forexample, for application as a vascular graft.

BACKGROUND OF THE INVENTION

It is known in the art to produce felts by applying pressure and heat toa tangle of threads. It has also long been known to produce multi-plyfelt by pressing bats of fleeces together, for example bats of differingquality as taught by U.S. Pat. No. 762,264 to Waring.

At least since the early 1990s thin webs of synthetic fibers have beenproduced by a technique known as extrusion fiber melt blowing. Theprocess basically involves extruding multiple strands through a line ofholes in a fiber melt blowing die, and allowing the parallel strands totangle while molten, to provide cohesion between the strands whencooled, forming the web.

Extrusion fiber melt blowing produces molten strands having adistribution of thicknesses that become randomly tangled. As the strandsare contact welded at tangle contact points, the resulting web generallyhas lower strength in one direction, i.e. the direction orthogonal tothe direction of extrusion. There are many applications of melt blownwebs that are applied to backings or other layers formed differently.

U.S. Pat. No. 6,048,808 to Kurihara et al. teaches a nonwoven fabric ofstretched filaments of polymers of different kinds, having a strengthequal to that of a woven fabric, and a method for manufacturing thesame. The fabric is characterized in that the nonwoven fabric isprovided with stretched filament webs comprising long filaments formedout of a plural kinds of thermoplastic polymers of different properties,the long filaments as a whole being aligned in one direction. The fabricmay be composed of threads that are spunbonded or melt-blown. Kuriharaet al. teach that a nonwoven fabric can be obtained by laminating websof different aligned directions. Both crosswise and obliquelyintersecting ways are applicable to laminating either longitudinallyaligned webs or transversely aligned webs. Kurihara et al. require websof different kinds of polymers be used, as does lamination, the methodof binding the webs, and the product resulting from the teachings ofKuirhara et al. does not have the useful properties described in thepresent invention.

U.S. Pat. No. 5,891,482 teaches a melt blowing apparatus for producing alayered filter media web product. A die apparatus wherein a layered webof melt blown fibrous filter media is produced by a unitary dieincluding several die sources with facing layers of the fibrous filtermedia being attenuated by opposed fluid streams at preselected includedangles and with the fiber layers being free from bonding together andwith the fibers in each layer being minimally bonded.

United States patent application publication number 2004/0035095 toHealey teaches a non-woven filter composite and a method for forming thecomposite. According to Healey, a cost effective, high efficiency, lowpressure drop, adsorptive, non-woven filter media is provided comprisinga high surface area synthetic microfiber, e.g., melt blown, fine fiberlayer. The filter media can also include one or more non-woven spun bondlayers and can be combined with a coarse fiber support layer. The coarsefiber support layer can itself be a low pressure drop syntheticmicrofiber, e.g., melt blown, layer adhered to a spunbond layer, and canserve as a prefilter to enhance overall performance.

Accordingly, it is known to produce a composite having a plurality ofdifferent non-woven layers.

Furthermore as taught in Journal of Materials Science 40 (2005)2675-2677 entitled “How to design a structure able to mimic the arterialwall mechanical behavior” to Jouan et al., it is known to producenon-woven mats of polypropylene fibers (6-14 μm in diameter), byapplying a heat treatment (15 min. at 120-150° C.) to a stack of 20 meltblown webs. Stress-strain curves of the mats produced by differenttemperatures of the heat treatment were compared with that of a femoralartery.

There remains a need for a non-woven mat that is inexpensive to produce,and has useful mechanical properties, including a uniform porosity.

SUMMARY OF THE INVENTION

Applicant has discovered that by subjecting a stack of differentlyoriented melt blown webs, or the like, to pressure and heat, a resultingmat can be produced. The mat will have desirable properties, such as auniform porosity, that is, a porosity having a narrow average sizedistribution, if the fibers of the mats are of a controlleddistribution. Specifically if at least a bottom 80% of the fiberdiameter distribution is substantially a normal distribution (i.e. has aregression coefficient (R) greater than 0.95, more preferably greaterthan 0.96, and more preferably greater than 0.97), and the standarddeviation and/or range of the bottom 80% of the fiber diameterdistribution is relatively small, and at least 15 webs are used, ahighly uniform porosity distribution results. For example, the standarddeviation may be less than 2 μm, more preferably less than 1.75 μm, morepreferably less than 1.5 μm, more preferably less than 1.4 μm, and/orthe range may be less than 5 μm, more preferably less than 4 μm, andmore preferably less than 3 μm, and more preferably less than 2.5 μm. Ifa desired pore size distribution is uniform and centered between 5 to 20μm, a mean fiber diameter of the bottom 80% of the fibers between about1.5 and 5.5 μm, more preferably 1.75 and 4.5 μm, and more preferably 2and 4.5 μm, is useful.

As melt blown webs are relatively inexpensive to manufacture, theconsolidation of these webs to produce mats having desirable propertiesconstitutes a highly cost-effective technique for producing mats.

The mat may have 15 or more layers, 16 or more layers, 18 or morelayers, 20 or more layer, 22 or more layers, 25 or more layers or 30 ormore layers. The mat may have less than 50 layers, or less than 40layers or less than 35 layers.

The webs may have surface densities of 0.1-10 g/m², or from 0.5-5 g/m²,or from 1-3 g/m².

The differences in orientations of two or more layers may be from about3° to 90°, from about 5° to 90°, from about 10° to about 90°, or fromabout 15° to about 90°.

The strand diameters of the mats may have average diameters ranging from200 nm to 300 μm, and has a chosen fiber diameter distribution.Generally the presence of 5-20% of fibers having strand diameters above(and possibly significantly above) the narrow distribution that accountsfor the vast majority of the fibers is not expected to significantlyalter the porosity either in terms of amount of porosity, or its sizedistribution. The lower bulk of the strand distribution (lower 80% forexample) that includes the finest and the vast majority of the strandsshould represent a more relevant property for determining the porosityattributes. The strand diameter distributions of the most uniformporosity mats produced are also marked as having substantially unimodal,and relatively smooth distributions, for at least the bottom 80% of thestrand diameters.

For cellular growth, a number of pores above the size of the cell shouldbe limited and a high porosity is required to permit fluid transportbetween the cells. For example, cells which have 10-20 μm diameters in asmallest direction (e.g. smooth muscle cells and endothelial cells) willrequire substantial porosity of the substrate, but will have to providepores small enough so that the cells can be supported. Given thisconstraint, it is desirable to have less than 20%, more preferably lessthan 15% and more preferably less than 10% of the fiber diameters above20 μm, and at least 40%, more preferably at least 45% of the fiberdiameters below 10 μm. This has been accomplished by controlling themean of the bottom 80% of the fibers to lie between about 1 and about 8μm, more preferably between 2 and 6.5 μm, and more preferably between 4and 5 μm. For example, if a porosity of about 10 μm is desired, a centerdiameter of around 3 or 4 μm (ranges of 2-6 μm, 2-4 μm) have been founduseful.

Applicant has further discovered that a method of producing a mat havinga high porosity of considerably uniform distribution is provided bypressing many webs. Uniformity of the porosity is achieved after acritical number of webs are applied. The specific number will vary withthe properties of the webs (polymer, density, fiber diameterdistribution, degree of orientation, etc.), but if polyethyleneterephthalate (PET) is used to produce the webs, and the webs havesubstantially all (80%) fibers with diameters between 2-7 μm, and webdensities of about 2.0 g/m², the number of webs is greater than about16, and a uniform distribution of pores ranging from 1-20 μm in diameterare provided. By choosing a number of webs above 16, and by selection ofthe orientations of the webs, a compliance (in given directions) andother parameters of the mat can be tailored to particular applications.The same web-forming process (extrusion melt blown) can be used togenerate PET webs having fiber diameters as low as 200 nm, andaccordingly a variety of other ranges of porosities can be providedusing this forming method.

Applicant has further discovered that mats produced with 15 or more suchwebs have a uniformity of porosity as well as a compliance that makethem good candidates for vascular grafts. The uniformity of the meanpore diameter within the range of 6 to 20 μm facilitates ingrowth ofcells including endothelial and smooth muscle cells desired for avascular graft. Advantageously, the cells can be seeded prior toimplantation.

Using the uniform porosity mat, Applicant has found that cell ingrowthcan be provided without the coating of the mat with extracellular matrixproteins such as gelatin. Furthermore human brain endothelial cells(HBEC) and smooth muscle cells (SMC) grown in the uncoated uniformporosity mats retain their specific phenotype, the ability to produceextracellular matrix proteins, such as collagen and elastin, andmaintain calcitonin-gene related peptide (CGRP) receptor expression andadenilate cyclase machinery for vasodilatation, suggesting that theapplication of the mats as grafts will not impair, but will facilitatecontinued vasodilatation within the grafted region of a blood vessel.Furthermore, both sides of a mat of the present invention can beindependently seeded with respective cells.

Conditions of the stacking of the webs may be chosen to select apreferred density of contact fused points, and a strength of the bondingof the contact points.

Accordingly a method of producing a mat is provided, the method beginswith producing a web of a tangle of strands of a polycondensationpolymer contact fused at tangle points. The strands have substantially asame mean orientation defining an orientation of the web, and thestrands have a diameter distribution such that at least a bottom 80% ofthe diameters are substantially unimodal and have a standard deviationof less than 2 μm. More preferably the at least the bottom 80% of stranddiameters have a standard deviation of less than 1.75 μm, and aregression coefficient measure of fit to a normal distribution of atleast 0.96. At least 15 sections of these webs are laid in a stack sothat the orientations of at least two web sections are substantiallydifferent, and then heat and pressure are applied to the stack of websto produce the mat, the heat being at a temperature above a glasstransition temperature and below a melting point of the polymer. The useof the same web to form the stack has numerous advantages for costeffective production. As the same web will have similar properties interms of sheet density, and mean fiber distribution, it will provideuniform densities within the mat. Herein sections of a web refers toparts of one or more web made from the same polymer using substantiallythe same web-forming apparatus and parameters.

The polycondensation polymer is preferably not highly hydrophobic, andmay be, for example, a polyester or a polycarbonate, for example. Morespecifically it may be one of: polyethylene terephthalate (PET),polycarbonate (PC), polytrimethylene terephthalate (PTT), and polylacticacid (PLLA), or combinations thereof. Most preferably PET, PTT, or PLLAis used.

The stacking of the webs, and distribution of the fiber diameters, arechosen to produce a desired pore size distribution. The pore sizedistribution may be strongly peaked, for example with 80% or 90% of thepores having a range of 10 μm. A pore size distribution having 90% ofthe pores having an average equivalent diameter less than 20 μm has beenfound beneficial for supporting cellular cultures.

Stacking the webs may involve applying groups of same orientation websections above and below differently oriented web sections, and mayinvolve substantially alternating orientations of groups of websections. The webs may be stacked on a mold.

A mat of uniform porosity may be achieved by stacking at least 15 webs.

Producing the web may involve extruding and stretching the strands,preferably using extrusion fiber melt blowing one or more webs andcutting the one or more webs to form the plurality of webs. Theextrusion fiber melt blowing may involve subjecting a molten tangledstrand output to a roller for providing a desired degree of orientationof the molten tangled strands.

A mat is also provided, the mat being formed from a plurality oforiented web sections of a same web stacked and compressed together,wherein the web is formed of substantially oriented, extruded strands ofa polycondensation polymer that is not highly hydrophobic, the strandsare contact-fused at tangle points, at least a bottom 80% of the stranddiameters are substantially unimodal and have a standard deviation with2 μm and the orientations of at least two of the web sections aresubstantially different.

The mat may be formed as a cylindrical or planar mat.

Accordingly there is also provided a vascular graft formed from acylindrical mat and having a compliance close to that of a blood vessel.The vascular graft may have a luminal inner wall seeded with endothelialcells, and/or an abluminal outer wall seeded with smooth muscle cells.The elastic modulus of the mat is comparable to that of a blood vessel.

Further features of the invention will be described or will becomeapparent in the course of the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

In order that the invention may be more clearly understood, embodimentsthereof will now be described in detail by way of example, withreference to the accompanying drawings, in which:

FIG. 1 is a schematic illustration of an extrusion fiber melt blowingapparatus;

FIGS. 2 a,b are schematic illustrations of stacks of webs for producingplanar and cylindrical webs respectively;

FIG. 3 is a schematic illustration of an apparatus for mat forming;

FIGS. 4 a-e are microscope images of five mats M1 . . . M5 havingdifferent webs and/or different numbers of webs;

FIG. 5 is an image of a typical cross-section micrograph of a mat;

FIG. 6 is a graph showing fiber size distribution, in terms ofcumulative percent of fibers as a function of diameter for mats M1 . . .M5;

FIGS. 7 a,b are graphs showing pressure-deformation curves resultingfrom cyclic pressure testing on cylindrical mats M5;

FIG. 8 is a graph showing pore size distribution, in terms of cumulativepore size percent as a function of average equivalent diameter of poresfor mats M1 . . . M5;

FIGS. 9 a-e are microscope images of the five mats M1 . . . M5 of FIGS.4 a-e at a magnification of ×500 instead of ×250;

FIGS. 10A,B,C,D,E,F and G are photo-micrographs of HBEC (A, C, D and E)and AoSMC (B, E, F and G) growing on mat M5, the mat serving as scaffoldfor the cells. HBEC (A) and AoSMC (B), stained with the vital dyeCFDA-AM, attached to the nonwoven mats with the body elongated followingthe direction of the fibers. HBEC labeled with fluorescein-conjugatedUEA-1 (green) (C) showed marked staining surrounding the plasma membrane(head arrow). HBEC immunofluorescently labeled with antibodies againstFactor VIII related-antigen (D) showed intracellular granular staining(arrow). HBEC stained with fluorescein-conjugated UEA-1 (green) andantibodies against type IV collagen (red) (E). Immunostaining of AoSMCwith antibodies against anti-smooth muscle α-actin (red) (F-G) andanti-elastin antibodies (green). Cellular nuclei were stained with DAPI(blue) (C-G);

FIG. 11 is a chart showing mean of fluorescent units of vital stainingwith CFDA-SE of HBEC grown for 6 days on uncoated (hatched bars) orgelatin-coated (solid bars) mats M1 . . . M5;

FIG. 12 is a chart showing mean of fluorescent units of vital stainingof AoSMC grown for 6 days on uncoated (hatched bars) or gelatin-coated(solid bars) mats M1 . . . M5;

FIG. 13 is a chart showing cAMP formation in response to differentconcentrations of CGRP by HBEC grown in mat M5.

DESCRIPTION OF PREFERRED EMBODIMENTS

The invention provides a mat formed from a plurality of oriented websstacked and pressed together, where each web is formed of substantiallyoriented strands, that are tangled and contact-fused at tangle points,such as the output of an extrusion fiber melt blowing apparatus. Theorientations of at least two adjacent webs are significantly different,and the webs are composed of a same material.

FIG. 1 schematically illustrates an extrusion fiber melt blowingapparatus. The blow melt extruder provides two channels, one for moltenpolymer, and the other for heated air, which meet at a point ofextrusion.

The extrusion fiber melt blowing apparatus shown has forced heated airsupply 1 in fluid communication with a manifold 2 which surrounds a die3, although this is not required. Basically an equal pressure onopposite sides is generally desired to reduce a likelihood of shearingthe molten strands. The heated air supply provides the air at a flowrate that is chosen to stretch the strands a desired amount less than abreaking point of the extruded strands, and at a temperature range thatdepends on a polymer extruded.

In accordance with the apparatus used by the Applicant in the examplesbelow, the heated air can be varied from about 280° C. to about 320° C.,resulting in a desired thickness and strength of the strands produced.Naturally the thickness and strength also depends on a draw rate of theextruder, and dimensions of the die, as will be understood by those ofskill in the art. The heated air serves to draw the fibers stretch them,and keep them in the molten state. It will be appreciated that differentpolymers may require more rapid cooling in order to provide contactfusing at tangle points and accordingly the heated air supply may besignificantly cooler or, in some embodiments, may be ambient or evenchilled air. Furthermore fluids other than air could be used to increaseor decrease the draw pressure on the exiting strands as may be requiredfor different polymers, or different dimensions of melt blow dieapertures. The die 3 has an egress directly above a driven roller 4.

A polymer material is fed into a hopper 6 where it is melted. Moltenpolymer is fed to the die 3 at a flow rate that is controlled andstabilized by a gear pump 5, which maintains a substantially constantfluid pressure of the polymer melt within the die 3.

The egress of the die 3, i.e. die tip 7 has a linear array of holesthrough which individual strands of the polymer melt are extruded, andvents for directing the heated air onto the exiting strands, as is knownin the art. For example, dies are known having 200 to 400 apertures ofdiameters ranging from 200 to 400 μm, and separations around 500 μm,with lengths of about 6-8 inches.

The passive roller 4 is positioned vertically and horizontally, anddriven to achieve desired degrees of tangling and orientation within theweb produced. Because of the heated air, the strands exiting the dieremain partially molten during contact with the roller 4. Accordingly,upon tangling, the tangle points are contact-fused and subsequentcooling results in the formation of a web. The roller 4 is positioned tomeet the strands a distance below the apertures, the distance beingchosen so that the polymer is cooled sufficiently before meeting theroller so that the molten strands do not puddle or pool on the roller,but not so cool as to have completely solidified, in which case littleor no contact-fusing will occur at tangle points (i.e. points at whichtangled strands touch). Naturally this distance depends on a temperatureof the molten polymer, a rate of extrusion, a temperature and volume ofthe hot air, thermal interaction of the air and molten strand, ambienttemperature, thickness of the strands, etc.

A horizontal position of the roller determines an angle that the strandsmeet the roller. This angle can vary between 0° and 180°. The horizontalposition and the rate of revolution of roller, and strand extrusion ratedetermine a dwell time of the strands on the roller, and a tensionapplied to the strands (apart from the pressure applied by the airsupply). The roller may be driven at different rates from 100 to 1000rpm and changing this rate within limits of stresses that can be appliedto the cooling strands, has an impact on the degree to which the strandsare tangled, and the degree to which they are oriented. Generally theslower the rotation, the less stress is applied to the strands by theroller, resulting in less elongation of the strands, and the longer thestrands dwell on the roller. The longer the dwell time the more tangledthe strands become, resulting in higher sheet/surface densities of theweb.

FIGS. 2 a and b schematically illustrate a planar stack and acylindrical stack of a few plies of the web. Each ply of the web isoriented at a substantially different angle from the previous ply, andthe angles between orientations of the first and subsequent webs varyfrom 0° to 90°. It will be appreciated that these webs can be stackedand/or cut by any applicable mechanical process.

FIG. 3 schematically illustrates a mat forming apparatus for applyingheat and pressure to a stack of webs. FIG. 3 shows an oven 10 containinga sealed evacuated bag 12 containing the stack of webs 13 held against asupport structure 14. The evacuation of the air within the bag causes acontinuous pressure to be applied across the surface of the stack,pressing the stack against the support structure. The temperature is ina range between a glass transition temperature and a melting point ofthe polymer, the temperature being closer to the melting point, the morecrystalline the polymer is.

Alternatively the web forming can be performed in an autoclave, as wellknown in the art. Autoclaves are designed to apply a wide range ofpressures and temperatures to a stack of webs for forming a mat. Thepressure and temperature required to consolidate the webs to a desireddegree naturally depends on the nature of the webs (density and fiberdiameter), and the nature of the polymer used.

Example 1 Materials

Polyethylene terephthalate (PET) was to produce non-woven webs inaccordance with these examples. The grade of PET used, Selar-pt 7086supplied by Dupont, has an inherent viscosity of 1, and was pretreatedby drying at 120° C. for at least 4 hours.

It will be appreciated by those of skill in the art that other meltextruded webs can be formed using polycarbonate (PC), or polyesters,such as polytrimethylene terephthalate (PTT), and polylactic acid (PLLA)among others. Applicant has also produced extrusion fiber melt blownwebs with polypropylene (PP). Specifically, the PP Valtec HH441 having amelt flow rate of 400 g/10 min and supplied by ExxonMobil was used.

It is known that the principles of melt blowing can be applied toproduce webs of other polymers if the thermal, and thermo-mechanicalproperties of the polymer melt are matched with the die, thermodynamicregime of the die and a correct fluid stream is used to draw/blow thestrands from the die.

Method of Web Formation

Webs (or veils) of non-woven fibers (i.e. strands) were produced using afiber melt blowing process. Three batches of webs were formed from PETusing slightly different processes: W1, W2, and W3.

The PET was introduced as pellets and melted at 312° C. A flow rate wasestablished by control of a gear pump, which fed a die having a blowmelt tip. The air was heated to 320° C. and a fan cycling at 1,000 RPMwas used to force the heated air through the manifold surrounding thedie, and out through vents adjacent line of holes in the die. Theextruded strands were directed onto a roller driven at 200 rpm. Theroller was 20 cm below the die tip, and met the roller (smooth steelcylindrical roller) at an angle of about 90°.

The die contained 230 aligned holes (about 300 μm in diameter). Air wasblown through a narrow gap 2 mm wide extending parallel to the line ofholes of the die. The air allowed for stretching of the fibers as wellas assisting in the drawing of the strands through the holes. Applicanthas achieved strand thicknesses varying from 200 nanometers minimumdiameter to 300 μm by changing the flow rate of the molten polymer (from12 kg/hr-0.5 kg/h) and the fan rate between 500 rpm and 1300 rpm.Examples of batch W1, W2 and W3 webs were produced with strand diametershaving the distribution shown in FIG. 6.

W1 webs were produced as follows: the PET flow rate was 2.05 kg/hr witha PET melt pressure of 225 Psi; the air temperature was 320° C., the fanwas driven at 1000 rpm; and the roller was driven at 200 rpm.

W2 webs were produced using the same apparatus, but were extruded at ahigher rate (the PET flow rate was 2.4 kg/hr with a melt pressure of 260psi), and the fan was driven at 500 rpm. The roller was again driven at200 rpm.

For W3 webs, the flow rate was 1.25 kg/hr, and the melt pressure 125psi. The air temperature was again 320° C., and the fan cycled at 1000rpm. The roller rotated at 105 rpm.

The same apparatus can be used to produce webs from PP, but useddifferent parameters. The flow rate of polymer was in the range of 4-12kg/hr, fan rate between 800 and 1300 rpm, the PP melt temperature was inthe range of 260-300° C. Furthermore the rolls were driven between 300and 800 rpm. Only ambient air was used for blowing the strands.

As the strand diameters of the webs are not expected to changesignificantly during the mat forming, the distributions of diameters ofthe strands of these webs can be determined from images of FIGS. 4 a-e,where 4 a-c are all produced from W1 webs having a density of 1.955g/m², 4 d is produced from W2 webs having a density of 1.731 g/m², and 4e is produced from W3 webs having a density of 1.924 g/m². It will benoted that in all mats except M4 at least 75% of the fibers havediameters ranging over less than 5 μm.

Methods of Mat Forming First Example

Both planar mats and cylindrical mats were prepared for mechanicaltesting. The planar mats were produced by stacking various numbers ofindividual webs onto a planar aluminum plaque. The webs were stacked byhand under minimal tension and trimmed to produce coupons of a size 20cm×20 cm for planar mats. The mat thickness depended on the number ofwebs stacked and was about 100 μm for 20 layers. The plaques were thenplaced in pressurized bags and, by evacuating the air in the bags, aneven vacuum pressure was applied to the coupons. The vacuum bag wasplaced in an oven at 90° C. for 30 minutes.

Second Example

Cylindrical mats, of about 30 cm in length, were produced on a 6-mmdiameter mandrel from 20 W1 webs with alternating orientations. Each webwas laid on the mandrel and cut so that edges of the web substantiallyabutted each other defining a seam. The seams of adjacent webs were notaligned to prevent a weakness from being defined throughout thecylindrical stacks. The mandrel was then inserted into an autoclave for30 minutes exposing the cylindrical stacks to 90° C. and a pressure of50 psi for 30 minutes.

Third Example

A third example of mat forming involved applying a slightly differentpressure and temperature regime, and was found to provide betterbonding. The temperature was marginally raised to 100° C., for aslightly shorter duration of 20 minutes, with under vacuum conditions(i.e. the mat is subjected to a pressure of about 14.7 psi).

These forming conditions were applied to produce cylindrical mats.Pressure was applied using a vacuum bag method which substantiallyreduced the defects caused by the wrinkling of the bag along the twoopposite sides of the bag where the inner surface of the bag meetsitself to form a seam. As will be appreciated, the pressure applied tothe mat along this seam is less, and there is a tendency for thepressure to nip the material rather than press it toward the mandrel.The pressure along the seam was significantly improved by surroundingthe mat with a thermoplastic film, which served as a bleeding layer forthe compression, improving the uniformity of the pressure applied to themandrel.

Other methods and systems for applying pressure and heat in a controlledmanner could equally be used to consolidate the webs to produce suchcylindrical mats.

Properties of Mats

Five kinds of planar mats were produced using webs of batch W1, W2 andW3: namely, mats M1 . . . M5 (please note these are respectivelyreferred to as structures A . . . E in the figures). Mats M1 . . . M3were made from W1 webs, and differ only in the number of webs in thestacks used to produce them. M1 mats were formed of 10 W1 webs, mats M2were formed of 15 W1 webs, and mats M3 were formed of 20 W1 webs. Ineach of these examples, the webs were oriented and adjacent webs werestacked with orthogonal orientations. Mats M4 and M5 were stacks of 20layers of W2 and W3 webs, respectively.

Plane micrographs of the five mats are presented in FIGS. 4 a-e. Amicrograph of a sectioned representative sample is shown in FIG. 5.While the illustrations are particularly of the mats produced using thefirst mat forming method, images of the mats made by the second andthird mat forming methods are equally well represented by the images ofFIGS. 4 b,c, and e, which are similar.

FIG. 6 shows a cumulative percent of fibers as a function of fiberdiameter. It can be clearly seen that the mats M1 . . . M3 have aboutthe same distribution (substantially within experimental errors). Thefiber diameters of M1 . . . M3 are mostly between less than 1 and about4 μm (more than 80% of the fibers, the next 10% are between 4 and 5.5μm, and the last 10% being between about 8-12 μm.

The mats having a second most uniform fiber diameter distribution are M5(80% of the fibers are between 2 and 6.5 μm, the rest ranging from 6.5to less than 10 μm).

The widest distribution of fiber diameters is present in mat D: between4 and 10 μm. In fact two populations are present in mat D: 40% between 3and 5 μm and 60% between 8 and 10 μm.

The measurements of fiber thicknesses were computed by image analysis on3 images from the plane of mats and 3 from the cross-section images ofthe mats. About 50 measurements were made for fibers diameterdistributions and 150 for pore size distributions (see FIG. 8).

Mats produced using the third method of web forming were experimentallyindistinguishable on these measures.

Mechanical Testing on Planar Mats

A variety of mechanical characteristics were obtained for planar matsmade by stacking W1 batch webs with various stacking patterns. Thestacking pattern is identified as follows: [0° _(a)/90°_(b)]_(c)-denotes a pattern that consists of a number (a) of W1 webs alloriented in parallel (at an angle of 0° with an axis) on top of whichare b W1 webs, all of which are oriented at an angle of 90° (althoughanother angle could be used) to axis, if a and/or b are absent, itrefers to a single web; and the number (c) that follows indicates anumber of repetitions of the pattern were stacked. An angle followingthe stack constitution indicates an angle at which the mechanicaltesting was performed, relative to the axis. The characteristics aresummarized in the Table 1:

TABLE 1 Compliances of Mats M1 . . . M5 Young's Yield modulus stressDeformation Deformation (MPa) (MPa) at Yield (%) at rupture (%)  [0°/90°]10-0° 252 ± 62 6.4 ± 1.0 5.5 ± 1.1  38 ± 12   [0°₂/90°₂]5-45°180 ± 15 4.1 ± 0.4 4.4 ± 0.4 40 ± 6   [0°₅/90°₅]2-45° 171 ± 17 3.9 ± 0.34.3 ± 0.3 32 ± 4  [0°₁₀/90°₁₀]1-45° 158 ± 20 3.5 ± 0.5 3.7 ± 0.3 21 ± 2

The tests were performed in tension mode using an Instronmicromechanical tester (Instron 5548) directly on coupons cut from themats. Testing was done at room temperature at a crosshead speed of 10mm/min according to testing conditions recommended in ASTM D638.Pneumatic grips with rubber faces were used to hold and pull thespecimen tested. Values of Young's modulus, yield stress and deformationat yield and deformation at rupture were obtained respectively from theinitial linear region, from deflection point at the end of first linearregion towards a flattened regime, and from final load decay on theload-extension slope.

It will be noted that a variety of compliance values can be obtained byvarying a number of layers and the orientations.

Mechanical Testing on Cylindrical Mats Example 1

Compliance measurements on about 15 cylindrical mats made according tothe second mat forming method (using 20 W1 webs) were performed on acustom-made test apparatus. This test apparatus consists of arod-mounted balloon connected to a pressurized nitrogen line via apressure regulator. The cylindrical mats were inserted onto therod-mounted balloon. Both applied pressure and the cylindrical matdiameter upon stretching were measured and logged. The pressure wasmonitored through a pressure transducer (AP-34K, Keyence Canada Inc.).The cylindrical mat diameter was measured using a laser scanner(LS-3100, Keyence Canada Inc.). The cylindrical mats tested were 5 cm inlength and 6.35 mm in diameter (unstretched).

The cylindrical mats were submitted to 100 pressure cycles between 0 and200 mmHg. Pressure was applied to the cylindrical mats at an approximaterate of 200 mmHg per sec. Pressure-diameter measurements were collectedat a rate of 10 points per sec. Measurements of compliance, C, were madefrom the pressure-diameter curves according to Equation 1:

$\begin{matrix}{C = \frac{\Delta \; D}{{D_{o} \cdot \Delta}\; P}} & (1)\end{matrix}$

where ΔD refers the variation of diameter when a variation of pressureΔP is applied to a cylindrical mat with an initial diameter D_(o). Theincremental modulus of elasticity, E_(inc), of the mats is calculated ina first approximation as its initial modulus of elasticity, given byEquation 2 [29]:

$\begin{matrix}{E_{inc} = \frac{D_{o}}{2 \cdot h \cdot C}} & (2)\end{matrix}$

where h is the thickness of the cylindrical mat. Assumptions were madethat the mat was uniform and cylindrical, and that it could beconsidered as an incompressible elastic thin wall tube.

The modulus of the cylindrical mat was measured using our custom-madeset-up. Pressure-diameter curves are shown in FIG. 7 a for cycles 1, 3,5, 10, 30, 50, and 100. These curves show that the cylindrical matexhibits an approximately constant compliance (initial slope) and thatit undergoes a slight permanent deformation (e.g., 0.05 mm/mm) uponpressure cycling, when passing from cycle 1 to 100. It will be notedthat the meaningful parts of the curve are the initial slope (after theinitial tension is applied to the cylindrical mat, until the pressurereaches its maximum), and that the remainder of the curves areschematic.

The average modulus obtained from Equation 2 for the 100 cycles is1.0±0.05 MPa.

Example 2

In an attempt to improve on the above experiment, the balloon wasreplaced with a balloon that had a more uniform opening mechanism thatdidn't present an artifact during the low pressure phase of the testing.The former balloon had a collapsing structure having folds runningspirally around the balloon, and therefore twisted during an initialpart of inflation.

The second example uses a cylindrical mat made with the third matforming method. The rest of the experimental details are the same asthat of the first radial testing example.

FIG. 7 b is a graph of pressure deformation curves during the increasingpressure phases of cycles 1, 5, 10, 30, 50, and 100, showing themeaningful parts of the curve. In comparison with the graph of FIG. 7 a,it will be noted that the stability of the mat throughout the cycling,which is inversely proportional to the displacement of the curves (inthe x direction) between the first and last cycles, is significantlyimproved. Specifically that about one fifth the displacement of the matsconsolidated as per the second forming method will be noted. At the sametime it is noted that the width of the curve was lower indicating astronger cohesion of the material and a smaller deformation in responseto an equal stress. Furthermore the linearity of the stress-strain curveis better, showing that the artifact attributed to the unfolding of theballoon in the earlier example is removed.

The average compliance calculated from Equation 1 for the 100 cycles is8.4±1.0×10⁻²%/mmHg and the average calculated modulus for the 100 cyclesis 1.7±0.2 MPa.

Burst Pressure Testing on Cylindrical Mats

The cylindrical mats made with the third mat forming method were firstsubjected to the 100 repeated inflating cycles to produce the pressuredeformation curves, and then was subjected to an applied pressure of 200mmHg continuously for 10 minutes without any additional changes indiameter (i.e any additional radial deformation) related to creep of thestructure. Then the pressure was increased until the structure failed,which is called the burst pressure. The burst pressure was 1325 mmHg.

Example 2 Porosity

The mats M1 . . . M5 are examined to determine a porosity distributionon the surface and throughout the mat. FIG. 8 is a graphicalrepresentation of results of porosity measurements performed on each ofmats. FIG. 8 plots the cumulative percent of pores as a function oftheir average equivalent diameter. The average equivalent diameter is adiameter of a circle having the same area as the pore at theintersection of a plane.

Mats M1 . . . M3 differ in the number of fiber stacks, which is expectedto have an effect on average pore size. This is clearly shown in FIGS. 4a-c: mat M1 (which has the least number of stacks) shows a distributionof pore size between 8 μm and 30 μm, mat M2: between 3 μm and 27 μm andmat M3: between 1 μm and 20 μm. The tightest distribution among these 3mats is thus mat M3. Mat M5 has a close distribution of pore size to matM3, minor differences are observed at the low end of the distribution,whereas mat M4 shows a large distribution of pore sizes with large pores(up to 50 μm).

It is noted that about 90% of the pores have a dimension less than 20 μmin M3 and M5, and that at least 50% of the pores have a dimensionbetween 5 and 17 μm. It will be noted that less than 20%, even less than15% and even less than 10% of the pores measured have pore sizes above20 μm, and further that more than 40%, and even more than 45% of thepore have sizes below 10 μm.

The narrow dimensional distribution of pores and the high porosity ofthese mats are features that make them potentially very useful for manyapplications. For example, a uniformity of pore size may be useful forcertain filters, such as air filters, or for keeping particles of agiven size in relative isolation from each other. About 90% of the poreshave diameters from 0-17 μm, and 80% are from 0-14 μm.

Prior Art Comparative Example Mats of PP

As taught by Journal of Materials Science 40 (2005) 2675-2677 to Jouanet al. referred to above, mats of PP were produced by stacking 20 pliesand applying a mat forming treatment comprising exposing the mats totemperatures of 120, 130, 140 and 150° C. It is not clear having regardto the paper, whether all the mats have a same orientation or whetherthe orientations alternate by 90° each time. Having regard to thereported 3-layer example having 50% orientation of fibers it would bereasonable to conclude that the 50% orientation must be an intrinsicproperty of the webs. Applicant has now obtained the specific webs usedand found that they are substantially oriented, and concludes that whatmust have been intended in the paper is that the webs were oriented withalternating layers orthogonal. Applicant concludes that the 3 layer dataexamples were interpolated based on samples having an even number oflayers. The paper indicates that these webs can be used to produce matsby changing orientations of the webs with respect to each other, toincrease a compliance of the mats, to improve a match with that of avascular wall.

The webs/veils used to prepare mats like those characterized by Jouan etal. were produced using polypropylene homopolymer (PPH, Valtec HH441having a melt index of 400 g/10 min and supplied by ExxonMobil) extrudedat a rate of 8 Kg/h. It is widely known that PPH is very hydrophobic.This presents numerous difficulties for many applications, and would beexpected to severely limit adhesion with cells, and cell growth.

Applicant has reproduced the mats described in Jouan et al. using thefollowing conditions. Four stacks of different numbers of webs ofalternating orthogonal orientations were laid: 20 [0°,90°]10, 2[0°,90°], 3 [0°,90°,0°] and 4 [0°,90°]2. Mats of each of these 4thicknesses were formed using a 0.5 mm thick frame-type mold placed in aCarver press (type F21123 model #M) under a pressure of 30 psi.

The extremes of the temperature conditions in the prior art paper (i.e.120° C. and 150° C.) were used to represent the variety of mats producedaccording to Jouan et al. In each pressing application, the pressure wasapplied for 10 minutes after the temperature was obtained to reproducethe results faithfully.

Table 2 schematically illustrates the average, maximum, minimum, andmedian fiber diameters within the resultant mats, as computed fromimages presented as FIGS. 9 a-e, as well as the standard deviation ofdiameter. It will be appreciated that the means of strand diameter ofeach sample is nominally the same, in that 14.9 μm is within thestandard deviation of each measure.

TABLE 2 Fiber distribution for 8 PRIOR ART PP examples Fiber 120° C.150° C. 120° C. 150° C. 120° C. 150° C. 120° C. 150° C. (μm) [0/90]₁₀[0/90]₁₀ [0/90]₁ [0/90]₁ [0/90/0]₁ [0/90/0]₁ [0/90]₂ [0/90]₂ ave 15.215.1 15.1 14.3 13.7 16.1 13.6 16.2 max 18.6 22.7 18.7 16.9 20.5 21.223.1 24.4 min 10.1 10.5 11.5 11.2 10.7 11.4 8.6 9.4 stdev 2.9 3.7 2.22.0 3.4 3.2 4.3 4.4 med 15.7 13.7 14.2 14.9 12.6 15.1 12.4 14.8

The diameters of the fibers of which the mats are composed, have adistribution between about 10 to about 20 μm. Jouan et al. asserts thatthe fiber diameters were 6-14 μm. By either account this distribution issignificantly broader (8-10) than the examples (mats M2, M3, and M5), ifyou remove the top 20% of the distribution. Specifically the range is8-10 μm, and the standard deviation is over 2 μm.

Table 3 lists the computed average, maximum, minimum and median porediameter of the two 20 ply mats. Naturally the porosities of the matshaving 4 or fewer webs were incomparable.

TABLE 3 Porosity distribution of PRIOR ART PP examples Pore diameteraverage maximum minimum std deviation median 120° C. [0°/90°]10 42.1109.5 8.2 19.6 36.6 150° C. [0°/90°]10 54.6 127.4 11.2 25.1 50.0

It will be noted that this pore distribution is broader than thatdefined by (mats M2, M3, and M5). Pores ranging from 10 to 100 μm havelimited applications as depending on the pore size, different retention,flow and other parameters are produced. Accordingly these examples donot demonstrate the advantages that can be achieved for producing anarrow porosity distribution, even if it had been produced of a polymersuitable for cell growth.

Without being limited to the foregoing theory in all aspects of theinvention, Applicant posits that the narrow distribution of porosityachieved in the invention is the result of a narrow, unimodaldistribution of the bottom 80% of the fiber diameters. Table 4 listsstatistical properties of the five mats M1 . . . M5 and the two examplesof PP mats formed at 120 and 150° C., respectively. The distributionsare for the entire range of fiber distributions.

TABLE 4 Fiber distributions comparison over entire distribution Fiber(μm) M2 M3 M5 M4 PP120 PP150 Min 2.33 2.16 2 3.43 10.14 10.51 Max 7.4812.21 11.3 9.72 18.58 22.72 Mean 3.7735 3.5475 5.095 6.9855 15.19215.146 Median 3.545 3.055 4.7 8.19 15.74 13.73 Std Dev 1.1557 2.15762.5496 2.4325 2.8583 3.6814

It will be noted that the distributions on the left (which producednarrow porosity distributions) and those on the right (which did notproduce narrow porosity distributions) are not very dissimilar.

TABLE 5 Fiber distributions comparison over bottom 80% of distributionFiber (μm) M2 M3 M5 M4 PP120 PP150 Average 3.3356 2.8631 4.0625 5.980714.594 13.715 Minimum 2.3300 2.1600 2.0000 3.4300 10.140 10.51 Maximum4.1900 3.8700 6.4000 8.7800 17.900 17.67 Median 3.3050 2.9100 4.50004.6050 15.730 13.41 Std Dev 0.55804 0.52147 1.3495 2.2363 2.6906 2.1345

Note that the bulk of the fibers in mats the left hand side have thebottom 80% fiber distribution with a standard deviation less than 2, andeven less than 1.4 μm. On the right hand side the fiber distributionsare considerably over 2 μm.

A general difference is also noted between the profiles of the fiberdiameter distributions of the right hand examples and left handexamples, in terms of how uniform they are. The fiber diameterdistributions were subjected to standard regression analysis, to measurethe fit of these values to a normal distribution centered on theaverage. The standard regression coefficients (R) are listed in thetable below, indicate a measure of fit of the data points to the normaldistribution centered on the respective median, are as follows:

TABLE 5 Unimodality of fiber distributions Fiber (μm) M2 M3 M5 M4 PP120PP150 R 100% 0.71753 0.90277 0.94901 0.90277 0.95441 0.95674 R 80%0.9878 0.97524 0.97229 0.89752 0.95104 0.98378

It is noted that the bulk (bottom 80%) in the left examples are highlyunimodal (i.e. R>˜96). M4, the PET mat that is more similar to M2, M3and M5 is significantly bimodal. PP120 does not significantly change itsregression coefficient depending on inclusion or exclusion of the top20%.

Example 3 Graft Application

It is noted that the average compliance and incremental modulus ofelasticity of the cylindrical mats produced for mechanical testingcompare well with reported E_(inc) values for external iliac (Blondel2000), carotid and abdominal (Learoyd 1966), radial wrist (Laurent 1994)and brachial (Bank 1995) arteries, which vary between 1 and 5 MPa. Thisdemonstrates that it is possible to reproduce the compliance of arterieswith nonwoven fiber mats. This compliance figure differs significantlyfrom the reported Dacron modulus values and expandedpolytetrafluoroethylene (ePTFE) shown in Table 6.

TABLE 6 Elastic modulus of graft polymers Elastic modulus Materials(based on compliance) Arteries* 1 to 5 MPa Dacron (woven structure) 2000to 4000 MPa ePTFE (porous membrane) 400-1800 MPa This work (avg. of 1.0± 0.05 MPa 100 pressure cycles)

The compliance matching of the graft with that of an artery, forexample, permits the graft to be implanted and would not requirereplacement at the rate of current grafts (such as Dacron and ePTFE),which may be formed of the same polymers, but are not as compliant,resulting in thrombogenicity.

For cellular growth, a number of pores above the size of the cell shouldbe limited. For example, cells which have 10-20 μm diameters in asmallest direction e.g. smooth muscle cells and endothelial cells willrequire substantial porosity of the substrate, but will have to providepores small enough to support the cell. Given this constraint, it isdesirable to have less than 20%, more preferably less than 15% and morepreferably less than 10% of the fiber diameters above 20 μm, and atleast 40%, more preferably at least 45% of the fiber diameters below 10μm. The porosity shown in FIG. 8 demonstrate that all of examples B, Cand E (i.e. M2, M3, and M5) have substantially this distribution, whichis believed to account for the remarkable adhesion and growth of thesecells on these mats demonstrated below.

Furthermore, as is known in the art, successful grafts integrate withthe existing blood vessels. Blood vessels have luminal cellular layerformed by endothelial cells (EC), and consequently it is desired toprovide grafts that permit continuity of the luminal layer. Likewise anabluminal layer of the blood vessels are formed with smooth muscle cells(SMC), and it is desirable to provide grafts that will permit continuityof the abluminal layer. Preferably the luminal and abluminal layers areformed with like cells to effectively provide continuation of the bloodvessel, for many functions including vasodilatation.

The surface pore size distributions of mats M1 . . . 5 look promisingfor the ingrowth of ECs and SMCs which would permit a graft formed ofthis non-woven fiber mat to retain and nourish ECs and SMCs. This couldbe performed by providing a temporary or permanent seal for the graftuntil the EC and SMC ingrowth is complete after implantation, butpreferably the graft is seeded with ECs and/or SMCs prior toimplantation.

Accordingly the mats prepared as described above were studied for theirresponse to culture of endothelial cells (EC) and smooth muscle cells(SMC). Fiber diameter distribution and pore size distribution arethought to have an effect on cell attachment and growth.

It is known in the art to functionalize grafts by applying compounds toimprove the adherence of cells and their proliferation. One of the mostcommon compounds used is gelatin. Accordingly, the tests for cellularattachment below are repeated with and without pretreatment of gelatin.

Cell Culture

Human brain endothelial cells (HBEC) were obtained from smallintracortical microvessels and capillary fractions (20-112 μm) harvestedfrom human temporal cortex excised surgically from patients treated foridiopathic epilepsy. Tissues were obtained with approval from theInstitutional Research Ethics Committee. HBEC were separated from smoothmuscle cells with cloning rings and grown in media containing Earle'ssalts, 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES),4.35 g/L sodium bicarbonate, and 3 mM L-glutamine, 10% FBS, 5% humanserum, 20% of media conditioned by murine melanoma cells (mousemelanoma, Cloudman S91, clone M-3, melanin-producing cells), 5 μg/mlinsulin, 5 μg/ml transferrin, 5 ng/ml selenium, and 10 μg/ml endothelialcell growth supplement (Stanimirovic et al., 1996). Cells were grown at37° C. in humidified atmosphere of 5% CO₂/95% air until about 80%confluence was reached. HBEC cultures were routinely characterizedmorphologically and biochemically. More than 95% of cells in culture:1-stained immunopositive for the selective endothelial markers:angiotensin II-converting enzyme and Factor VIII-related antigen;2-incorporated fluorescently labelled Ac-LDL; and 3-exhibited highactivities of the blood-brain barrier-specific enzymes:γ-glutamyltranspeptidase and alkaline phosphatase (Stanimirovic et al.(1996) J Cell Physiol 169: 455-467).

Human aortic smooth muscle cells (AoSMC) were obtained from Clonetics(Walkersville, Md., USA) and cultured in smooth muscle growth media(SmGM®-2 BulletKit®, Clonetics) at 37° C. in humidified atmosphere of 5%CO₂/95% air until reach about 80% confluence. More than 95% of the AoSMCin cultures stained immunopositive for the selective smooth musclemarker α-actin.

HBEC and AoSMC were respectively trypsinized with 1 ml of either GRP-2(Cells systems) or GIBCO (0.25%) trypsin. The mats M1 . . . M5 werepunched into discs, and the discs were placed on Falcon 24-well platesand pre-wetted overnight either with cell media or with 0.5% gelatin. 50μl of culture media containing 10⁵ cells were applied to the meshes andlet sit for 20 min before filling the wells with additional 450 μl ofmedia. Cells were allowed to grow on the meshes for 6 days at 37° C. inhumidified atmosphere of 5% CO₂/95% air.

HBEC and AoSMC Staining

To visualize and quantify the number of living cells on the mats (planardiscoidal scaffolds), cells were washed twice with warm Hank's balancedsaline solution (HBSS), incubated with 10 μg/ml a vital dye called CFDA™(Molecular Probes, Invitrogen Corp.) at 37° for 45 minutes and thenwashed again with HBSS. Fluorescence of the cells grown both on themeshes and on the bottom of the well (after removing the meshes) wasmeasured using a cytofluorimeter plate reader (Bio-Tex FL600) at 485 nmex/530 nm em.;

HBEC and AoSMC grown on uncoated scaffolds were fixed with 4%paraformaldehide for 10 min at room temperature (RT). Cells were thenrinsed 4 times with HBSS, and permeabilized with 0.1% Triton™ X-100 (EMScience Gibbstown, N.J., USA) in HBSS at RT for 10 min. After rinsing,cells were blocked with 4% serum in HBSS for 1 h at RT. HBEC were thenincubated with mouse anti collagen IV primary antibody (1:500 dilution,Abcam, Cambridge, Mass., USA) while AoSMC were incubated with mouseanti-smooth muscle a-actin primary antibody (1:500 dilution, R&Dsystems, Minneapolis, Minn., USA) and rabbit anti-elastin primaryantibody (1:500, Ciderlan, Hornby, ON, Canada) for 1 h at RT. Cells wererinsed twice with HBSS, and HBEC were exposed to both goat-antimouseAlexa™ Fluor 568 secondary antibody (1:500 dilution, Molecular Probes,Burlington, ON, Canada) and Fluorescein Ulex Europaeus Agglutinin-1(Vector Lab. Inc., Birlingame, Calif., USA) while AoSMC were exposed togoat anti-rabbit Alexa Fluor 488 secondary antibody (1:500 dilution,Molecular Probes) and goat anti-mouse Alexa Fluor 568 secondary antibody(1:500 dilution) for 30 min at RT. Cells were rinsed twice with HBSS andcovered with DAKO mounting media spiked with DAPI (2 μg/ml, SigmaAldrich, Oakville, ON, Canada) and placed on glass slides.

Microphotographs of the cells attached to the mats were taken using anOlympus 1X50 microscope. Images were captured using a digital videocamera (Olympus U-CMT) and analyzed with Northen Eclipse v. 5.0software.

HBEC (FIG. 10A) and AoSMC (FIG. 10B) stained with the vital dye CFDA-AMshowed attachment to the mats. The cells were elongated and alignedfollowing the direction of the fibers (FIG. 10A-B). After 6 days, themat appeared completely populated with cells that spread through thepolymer (FIG. 10C-E) and retained their specific molecular markers; HBECexpressed factor VIII (FIG. 10C) and bound Ulex Europaeus I lectin (FIG.10D-E) and AoSMC expressed a-actin (FIG. 10E-G). In addition, HBECexpressed collagen IV (FIG. 10E), an ECM that provides stiffness to thevascular wall and AoSMC expressed elastin (FIG. 10G), an ECM responsiblefor elastic recoil of arteries.

The fact that some control over a degree of fiber orientation isprovided by controlling a roller speed and position relative to theother parameters of the extrusion fiber blow melt web formation,presents the possibility of allowing the design of the mats with optimalweb orientation to minimize risks of thrombogenicity. For exampleluminal webs oriented with the fibers aligned in the direction of thevascular long axis and to maximize smooth muscle contractibilityabluminal webs may be orientated with the fibers alignedcircumferentially in the direction of the short axis.

Because the mats are composed of fibers, there are networks ofporosities running through them. The network-porosity of these matssuggest that communications paths and fluid channels would be providedbetween ECs and SMCs in a similar manner to how they are provide by thematrix of elastin and collagen fibers that form the media, andconsequently will facilitate integration of the graft within thesewalls.

Both HBEC and AoSMC preserved their specific cellular phenotype whenseeded on mat M5 (i,e, structure E). HBEC retained both the expressionof Factor VIII-related antigen [Hormia et al. (1983) Experimental CellResearch 149(2):483-497.], a classic marker of endothelium that formscomplexes with Von Willebrand factor (vWF), and the binding capacity toUlex Europaeus I agglutinin, a lectin that selectively recognizesL-fucose moieties of multiple glycoproteins present on the surface ofendothelial cells [Hormia et al. (1983) Experimental Cell Research149(2):483-497; Holthofer et al. (1982) Laboratory Investigation; AJournal of Technical Methods and Pathology 47(1):60-66]. AoSMC alsomaintained their capacity to express smooth muscle alpha actin, aselective marker of vascular SMC that has been shown to be regulated byhormones, cell proliferation, and altered by pathological conditionsincluding oncogenic transformation and atherosclerosis [Chaponnier andGabbiani (2004) The Journal of Pathology 204(4):386-395].

The elastic properties of large arteries and their capacity tosynthesize vasoactive substances are key elements for the ability of thearterial wall to function as a modulator of blood pressure andcardiovascular hemodynamics. The passive biomechanical properties of thearterial wall are influenced predominantly by the extra-cellular matrix(ECM) proteins, collagen and elastin. Collagen provides the tensilestiffness for the resistance against rupture while elastin dictates theelastic properties of the blood vessels. Combined with collagen, elastinprevents irreversible deformation of the vessel against pulsatile flow[Faury (2001) Pathologic-biologic 49(4):310-325]. Alterations incollagen and elastin vascular content have been associated topathological conditions such as artery stiffening and resistance arterynarrowing, two key features that contribute to the development ofhypertension [Arribas et al. (2006) Pharmacology & Therapeutics111(3):771-791]. HBEC and AoSMC growing on the mats expressed collagenand elastin, respectively, indicating that the cells not only preservetheir phenotype but they also retain their capacity to produce ECMproteins essential to maintain EC-SMC communication and modulatevascular cellular functions such as cell proliferation, adhesion andmigration [Brooke et al. (2003) Trends in Cell Biology 13(1):51-56].

FIGS. 11 and 12 are charts showing fluorescence emitted by the cellsstained with the vital dye CFDA-AM. Bars represent fluorescence units oflight emitted at 530 nm under excitation at 485 nm. The uncertainty wasderived from standard deviations of 2 experiments performed intriplicate. FIG. 11 represents HBEC cell populations, whereas FIG. 12represents AoSMC populations on M1 . . . M5. The solid bars refer tosamples that were presoaked in gelatin, and the cross-hatched bars referto non-pretreated mats (soaked only in cell culture). Surprisingly themats M1 . . . M5 were better designed to hold the cells without theassistance of gelatin.

cAMP Production

As is also known, calcitonin gene related peptide (CGRP) is one of themost potent vasodilators. Its vascular effect is mediated by activationof adenylate cyclase and the subsequent formation of cAMP in smoothmuscle and endothelial cells. Accordingly cAMP production is a measureof the ability of the EC seeded on the mats to respond to vasodilatoragents such as CGRP.

The effect of CGRP in the production of cAMP was assessed in HBECgrowing on mat M5. The cells were incubated (10 min) with increasingconcentrations (0.5-1 μM) of CGRP in phosphate buffer saline containing0.2% bovine serum albumin and 1 mM 3-isobutyl-1-methyl-xanthine. Levelsof cAMP were determined with a commercial enzyme immunoassay kit(Biotrak, Amersham). The cells were dissolved in 0.1 NaOH and proteincontent measured by Lowry's method. cAMP levels were expressed as afunction of protein content in cell extracts.

The results shown in FIG. 13 indicate an increase in cAMP production inresponse to increasing concentrations of CGRP.

The active biomechanical properties of the arterial wall depend on theactivation of vascular SMC either directly or through endothelialcell-dependent mechanisms. CGRP is a potent dilator of human brainarteries [Moreno et al. (2002) Neuropharmacology 42(4):568-576], ˜100 to1000 times more potent than other vasodilators such as adenosine, SP, oracetylcholine. It acts through activation of type II G-protein-coupledreceptors located in both EC and SMC and posterior stimulation ofadenylate cyclase activity [Moreno et al. (2004) Encyclopedia ofEndocrinology and Endocrine Diseases (Academic Press. Elsevier Inc.)1:421-435]. Reduction of CGRP release from nerve terminals anddown-regulation of CGRP receptor expression in vascular tissues havebeen reported to be involved in the pathophysiology of hypertension[Deng and Li (2005) Peptides 26(9):1676-1685]. EC seeded on mat M5responded to CGRP producing similar levels of cAMP than those producedby EC seeded on gelatin-coated plastic discs. This indicates that theinteraction of EC with the scaffolds neither alters the CGRP receptorexpression nor the dilatory capacity mediated by cAMP formation of theEC.

In summary, in this study, nonwoven PET fiber mats were developed usinga melt blown process, which were used as vascular scaffolds. Thisfabrication method allowed the control of fiber diameter, structuralporosity and mechanical compliance. Following an iterativemanufacturing/cell culture evaluation process, mats were obtained havingporosity and fiber diameters required to produce good scaffolds. BothHBEC and AoSMC were able to proliferate and spread in the scaffolds,retained their characteristic cell phenotype (HBEC: factor VIIIexpression and Ulex Europaeus I lectin binding; AoSMC: α-actinexpression) and respectively produced collagen and elastin. HBEC alsopreserved the ability to produce cAMP when stimulated with CGRP, a verypotent vasodilator agent. Mechanical testing of the cylindrical matsshowed compliance and deformation ranges similar to native arteryvalues. In contrast, commercial woven or knitted PET fiber or ePTFEgrafts have a 10-fold lower compliance. All together this indicates thatthe biomechanical and biocompatible characteristics of this novelscaffold are applicable as vascular grafts.

In conclusion, the mats M2, M3 and M5 present promising characteristicsfor vascular graft fabrication such as: controllable porosity, fiberorientation and compliance; no toxicity; good HBEC and AoSMC cellattachment and proliferation following fiber alignment; the retention ofAC machinery for vasodilation of HBEC grown thereon; the comparabilityof elastic moduli of the mats with that of native arteries (˜1 MPa),especially in comparison with those of Dacron or PTFE; and finally thelow cost of manufacture.

Other advantages that are inherent to the mats are obvious to oneskilled in the art. The embodiments are described herein illustrativelyand are not meant to limit the scope of the invention as claimed.Variations of the foregoing embodiments will be evident to a person ofordinary skill and are intended by the inventor to be encompassed by thefollowing claims.

1-21. (canceled)
 22. A vascular graft comprising a mat formed as acylinder, wherein the mat has a compliance close to that of a bloodvessel, and wherein the mat is formed from 15 to 50 web sections of asame web stacked and compressed together, wherein: the web is formed oforiented, extruded polymer strands contact-fused at tangle points, thestrands in a given web section being parallel to one another therebydefining an orientation of the given web section, the orientations of atleast two web sections differing by an angle between 3° and 90°; and atleast the smallest 80% of strands have a mean strand diameter between 2and 4.5 μm with a standard deviation within 2 μm and strand diametersare unimodal with a regression coefficient measure of fit to a normaldistribution of at least 0.96.
 23. The vascular graft according to claim22 wherein a luminal inner wall of the mat is seeded with endothelialcells, and/or an abluminal outer wall is seeded with smooth musclecells.
 24. The vascular graft according to claim 23 wherein the innerwall of the cylindrical mat is covered with ingrown endothelial cellsthat retain their characteristic cell phenotype, and/or the outer wallis covered with ingrown smooth muscle cells that retain theircharacteristic cell phenotype.
 25. The graft of claim 22 wherein the matis formed of a hollow cylinder having an average compliance between7.4×10⁻²%/mmHg and 9.4×10⁻²%/mmHg.
 26. The graft of claim 22 wherein thepolymer comprises a polycondensation polymer.
 27. The graft of claim 26wherein the polycondensation polymer is one of: polyethyleneterephthalate (PET), polycarbonate (PC), polytrimethylene terephthalate(PTT), and polylactic acid (PLLA), or a combination thereof.
 28. Thegraft of claim 22 wherein the polycondensation polymer is a polyester,and the orientations of adjacent web sections are orthogonal.
 29. Thegraft of claim 22 wherein the web is produced of PET strands and has aporosity network with about 90% of the pores having a dimension lessthan 20 μm.
 30. The graft of claim 29 wherein at least 50% of the poreshave a mean diameter between 5 and 17 μm.
 31. The graft of claim 25wherein: (a) the polymer comprises a polyester; (b) the orientations ofadjacent web sections are orthogonal; and, (c) the web has a porositynetwork with about 90% of the pores having a dimension less than 20 μm.32. The graft of claim 25 wherein: (a) the polymer comprisespolyethylene terephthalate; (b) the orientations of adjacent websections are orthogonal; and, (c) the web has a porosity network withabout 90% of the pores having a dimension less than 20 μm, and whereinat least 50% of the pores have a mean diameter between 5 and 17 μm.